N-acetyl-L-cysteine (NAC) has been used to treat acetaminophen overdose since the 1970s and we already know that it has an excellent safety profile from these many decades of experience. NAC has also been used to treat patients with neurological conditions. For example, NAC was found to facilitate aspects of cognition in Alzheimer's disease. Furthermore, NAC was found to help resolve symptoms of blast injury in soldiers. However, questions have arisen about the low bioavailability of NAC and the need for high doses to penetrate the brain. We will develop use of the cell-permeable derivative, NAC ethyl ester (NACET), in a novel ?-synuclein fibril-based model of Parkinson's disease. We will also establish the mechanism of protective action at the molecular level. Our pilot data on NAC already suggest that NAC can protect neurons and astrocytes in a heat shock protein-dependent manner. That is, NAC raises Hsp70A1/2 and pan-inhibitors of Hsp70 abolish its protective effects. This novel mechanism of NAC action has not been described by others in the literature, but may help explain why NAC has been effective in multiple double blind, placebo-controlled clinical trials. Here we will test the hypothesis that NACET can protect olfactory bulb neurons against ?-synucleinopathy in an Hsp70-dependent manner. We have chosen to examine the olfactory bulb in these studies, as bulb atrophy and cell loss and olfactory dysfunction develop early in the course of Parkinson's disease. Aim 1a) Treat primary olfactory bulb neurons with a-synuclein fibrils and various concentrations of NACET. Determine if NACET protects against cell loss, raises Hsp70 family members, and mitigates an increase in ubiquitinated proteins and in Lewy-like inclusions. Aim 1b) Use 3 independent Hsp70 inhibitors to test if NACET-mediated protection is abolished with loss of Hsp70 activity. Aim 1c) Test if NACET raises Hsp70 levels by activating the transcription factors Nrf2 and HSF1. If Nrf2 or HSF1 is translocated to the nucleus after NACET treatment, knock it down with RNA interference to see if NACET-mediated protection is abolished. Aim 2a) Infuse ?-synuclein fibrils into the mouse olfactory bulb and administer NACET in drinking water. Count NeuN+ neurons and Lewy-like inclusions by stereology after 6 months. Aim 2b) Repeat the fibril injections and NACET treatments and collect mouse tissue for Western blotting. Measure levels of Hsp70 family members and ubiquitinated proteins. Test the hypothesis that NACET will reduce neuron loss and Lewy-like inclusions in vivo and that it will raise Hsp70A1/2 and lower ubiquitinated proteins in olfactory bulb tissue. NACET may be the first treatment to safely boost Hsp70 defenses and reduce protein-misfolding stress in humans, with the potential to revolutionize Parkinson's treatment. These studies exemplify bedside-to-bench research, where compounds that are known to work in humans are examined post hoc in experimental models to yield deeper insights into disease etiology and drug action. This approach has been successful in the field of psychiatry for many years and it is time to leverage it in the battle against Parkinson's disease.